Planta (Berl.) 125, 201--211 (1975) 9 by Springer-Verlag 1975
The Control of Glutamine Synthetase Level in Lemna minor L. David Rhodes, G. A. Rendon, and G. R. Stewart Department of Botany, The University, Manchester. M13 9PL, U.K. Received 10 may; accepted 21 May 1975 Summary. The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mlV[ ammonia and 5 mM glutamine as nitrogen source, lead to a 4-5 fold reduction of the maximum activity measurable on 5 mM y-aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutaminc. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.
Introduction The conversion of glutamic acid to glutamine is generally regarded as a key step in the intermediary nitrogen metabolism of micro-organisms (see e.g. Brown et al., 1974). The reason for this lies in the fact t h a t glutamine can serve as a nitrogen donor in the biosynthesis of several nitrogenous metabolites which are precursors of proteins and nucleic acids. I n view of this key role of glutamine it is not surprising t h a t the enzyme catalysing its synthesis is controlled b y a variety of complex regulatory mechanisms. The glutamine synthetase of Escherichia coli is in fact regulated b y several mechanisms including repression and derepression of its synthesis, cumulative feedback inhibition and rapid reversible inactivation (Shapiro and Stadtman, 1970). Repression of glutamine synthetase has been reported in other microorganisms, including Anabaena cylindrica (Dharmawardene et al., 1973). Bacillus subtilis (Rebello and Strauss, 1969), Candida utilis (Ferguson and Sims, 1974a), KlcbsieUa aerogenes (Meers and Tempest, 1970), Lactobacillus arabinosus (Ravel et al., 1965), Neurospora crassa and Aspergillus nidulans (Pateman, 1970) and in Saccharomyces cerevisiae (Kohlaw et al., 1965). I t is often considered t h a t ammonium is the co-repressor of enzyme synthesis (see Shapiro and Stadtman, 1970), although glutamine appears to have this role in Lactobacillus and Candida. I n contrast to repression control, the rapid inactivation by adenylation of the enzyme, first described in E. coli, appears to be restricted to certain members of the Gram-negative bacteria (Gancedo and ttolzer, 1968). However an alternative mechanism of rapid inactivation has been reported in several species of yeast (Ferguson and Sims, 1971). I n higher plants less is known of the metabolic function of glutamine although it acts as a nitrogen donor in the biosynthesis of asparagine (Streeter, 1973) 1 Planta (Berl.)
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a n d t r y p t o p h a n e (Widholm, 1974). R e c e n t l y a more stategic role for g l u t a m i n e i n higher p l a n t s has emerged with t h e discovery of a g l u t a m i n e d e p e n d e n t g l u t a m a t e s y n t h a s e (Dougall, 1974; Lea a n d Miflin, 1974). I n a d d i t i o n to these metabolic functions, g l u t a m i n e is i m p o r t a n t i n the storage a n d t r a n s p o r t of n i t r o g e n i n higher p l a n t s (Pate, 1973). A l t h o u g h the g l u t a m i n e s y n t h e t a s e (E.C. 6.3.1.2) of higher p l a n t s appears to be sensitive to a form of feedback i n h i b i t i o n similar to t h a t i n bacteria (ttaystead, 1973; K i n g d o n , 1974; Rhodes a n d Stewart, 1974), there is a lack of i n f o r m a t i o n available on the possible r e g u l a t i o n of its synthesis. W i t h these considerations i n m i n d , the regulation of g l u t a m i n e s y n t h e t a s e level i n the higher p l a n t L e m n a m i n o r has been studied.
Materials and Methods Organism and Growth Conditions. The strain of Lemna minor L. and the basal growth medium employed in the present study were those described previously (Stewart, 1972a). Plants were grown at 26~177176 under conditions of continuous illumination (30001ux). With the exception of glutamine, which was sterilised by millipore filtration, all other nitrogen sources were added to the medium immediately prior to autoclaving. The medium referred to as amino acid mixture was based on the composition of the pool of free amino acids in Lemna and contained the following concentrations of amino acids: alanine 0.37 raM, arginine 0.05 mM, aspartie 0.48 raM, cystine 0.005 raM, glutamic 0.86 raM, glycine 0.33 raM, histidene 0.025mM, isoleueine 0.13raM, leueine 0.14mM, Lysine 0.05raM, methionine 0.01 mi~, phenylalanine 0.045 raM, proline 0.03 mM, serine 0.58 raM, threonine 0.14 mM, tryptophane 0.01 raM, tyrosine 0.04 mM, and valine 0.06 mlVI. Growth Studies. Growth rates were determined using 100 ml of the appropriate medium with an initial innoculum of 10-15 fronds. Frond number was counted daily and the time for frond number to double was determined by regression analysis of log frond number versus time. Ammonia and Amino Acid Pool Analyses. These were extracted and determined as described previously (Orebamjo and Stewart, 1974). Enzyme Extraction and Assay. The enzyme extraction procedure was that described previously by Stewart (1972a). The optimum extraction buffer for the determination of glutamine synthetase activity was 0.05 M Imidazole-HC1, pI-I 7.2, containing 0.5 mM EDTA and 1.0 mM dithiothreitol. The reaction mixture for the determination of synthetase activity contained: 36 fzmol ATP, 90 tzmol MgS04, 12 Izmol hydroxylamine, 184 tzmol L-glutamate, and 100[zmol Imidazole-HC1. The final volume of the reaction mixture was 2 ml and had a pI-I of 7.2. The reaction was initiated by the addition of enzyme extract and was incubated at 30~ for 30 rain. The reaction mixture for the determination of the transferase activity contained: 0.34 Izmol ADP, 5 ~mol MnCI2, 34 Izmol hydroxylamine, 130 tzmol L-glutamine, 66 ~mol sodium arsenate and 200 tzmol Tris-acetate, pI:i 6.4 (final volume 2 ml). The reaction was initiated by the addition of enzyme extract and was incubated at 30~C for 10 rain. tIydroxymate was determined by the addition of ferric chloride reagent (Ferguson and Sims, 1971). Glutamyl Hydroxymate was Used as the Standard. Glutamine synthetase activity was determined in vivo as described by I~hodes and Stewart (1974). Plants were permeabilised by six cycles of freeze-thaw treatment followed by vacuum infiltration with the reaction mixture. The substrate concentrations were those used in the in vitro assays. Protein precipitated from the enzyme extracts with 10% triehloroacetic acid was determined as before (Stewart, 1972a). Total protein was determined by incubating plants in 60% ethanol overnight at room temperature to remove soluble Folin positive components. After a further 48 hrs. incubation in 0.72 M 1NaOH, the total protein content of the filtrate was determined as above. I n vitro enzyme activity is expressed as nmol or fzmolhydroxymate/m/mg extractable protein. I n vivo activity is expressed as tzmol/m/mg total protein. Total Nitrogen. The total nitrogen content of the plants was determined as described by Stewart et al. (1973).
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Table 1. Effect of nitrogen source on glutamine synthetase level, growth rate and total nitrogen content Glutamine synthetase (in vitro activity). Nitrogen source (5 re_M)
Synthetase (nmol/m/ mg protein)
Transferase (~zmol/m/ mg protein)
Doubling time (h)
Total nitrogen (% of dry wt)
Alanine y-Aminobutyrate Amino acid mixture a Ammonium chloride Ammonium nitrate Aspartate Asparagine Glutamate Glutamine Glycine Potassium nitrate
57 133 89 56 46 73 61 70 42 58 98
1.2 2.2 1.6 1.1 1.0 1.5 1.4 1.4 0.8 1.2 1.7
56.0 59.2 43.0 33.0 36.6 42.5 34.0 45.4 46.0 69.0 36.0
4.6 3.8 3.2 8.1 7.7 3.1 10.2 3.4 7.6 4.7 6.1
Results are the average of at least three separate determinations. Plants were adapted to different nitrogen sources through at least two sub-cultures before analysis. a Amino acid mixture total concentration 3.4 raM.
Results The specific activity of glutamine synthetase varies considerably depending on the source of nitrogen that L e m n a is grown on (Table l). Maximum activity of the enzyme was observed in plants grown on y-aminobutyrate and nitrate. The activity in plants grown on glutamine is some 3-4 fold lower than that in y-aminobutyric acid grown plants. Ammonium grown plants have a low specific activity of the enzyme compared with nitrate grown plants, but their activity is higher than that of glutamine grown plants. This pattern of variation in glutamine synthetase level is similar to that reported in many micro-organisms. The variation in specific activity with respect to nitrogen source is similar whether measured by the synthetase or transferase assay. There is no evidence for any marked change in the ratio of transferase to synthetasc activity. A comparison of enzyme level with either growth rate or nitrogen content indicates that there is no simple relationship between growth (or nitrogen status) and enzyme level. I n the observations described above, all nitrogen sources were present at a concentration of 5 raM. The relationship between enzyme level and the concentration of various nitrogen sources is shown in Fig. 1. The response to increasing concentrations of ammonium ions is striking, with there being a 50% reduction in specific activity as the concentration is increased from 0.1 to 5 raM. I n contrast, increasing the nitrate concentration has little effect on the glutamine synthetase level. Increasing the concentration of glutamate in the medium results in a small reduction in enzyme level. The pattern of response to increasing concentrations of the nitrogen source is similar whether measured by the in vitro or in vivo assay procedures.
204
D. Rhodes et al. A
B 1.0
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0.25
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i
r
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Fig. 1. Effect of nitrogen source concentration on the specific activity of glutamine synthetase (transferase) in vitro (A) and in vivo (B). Activity is expressed as ~mol/m/mg protein. Potassium nitrate (I), Glutamate (A), Ammonium chloride (e)
The results in Table 1 and Fig. 1 suggest that the glutamine synthetase level could be regulated by the endogenous pool of either glutamine or ammonium ions. In order to discriminate between these two possible effector molecules, plants have have been grown under conditions where the pools of ammonium and glutamine can be independently altered. Nitrate adapted plants are characterised by a low pool of both glutamine and ammonium (Orebamjo and Stewart, 1974). Under such conditions one can predict that the accumulation of glutamine is likely to be limited by the availability of ammonium ions. Ammonium adapted plants have a high pool of glutamine and ammonium (Orebamjo and Stewart, 1974) and under these conditions glutamate availability is more likely to limit the further accumulation of glutamine. I t is predictable then that the addition of glutamate to ammonium adapted plants should bring about an increase in the glutamine pool and a decrease in the pool of ammonium. The addition of glutamate to nitrate grown plants should not however result in any marked increase in the glutamine pool, due to the low availability of ammonium. The addition of glutamine to either ammonium or nitrate grown plants should bring about an increase in the pool of glutamine independent of the availability of ammonium ions. Accordingly, the response of nitrate and ammonium adapted plants to the addition of glutamate and glutamine have been determined. The effect of various concentrations of these nitrogen sources on the glutamine synthetase level of 5 mM nitrate and 5 mM ammonium grown plants is shown in Figs. 2 and 3. 5 mM glutamate brings about a 20% reduction in the enzyme level of nitrate adapted plants and a much more pronounced lowering of activity of ammonium
Control of Glutamine Synthetase Level
205
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Fig. 2. Effect of various concentrations of Glutamate on the specific activity of glutamine synthetase of 5 mM Nitrate grown plants (i), and 5 mM Ammonium grown plants (o). A: Synthetase activity (nmol/m/mg protein). B: Transferase activity (~mol/m/mg protein). Activities were determined by the in vitro procedure
10C
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Fig. 3. Effect of various concentrations of Glutamine on the specific activity of glutamine synthetase of 5 mM Nitrate grown plants (i), and 5 mM Ammonium grown plants(o). A: Synthetase activity (nmol/m/mg protein). B: Transferase activity (~mol/m/mg protein). Activities were determined by the in vitro procedure
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Table 2. Effect of nitrogen source on soluble nitrogen pool and glutamine synthetase level Nitrogen source (5 mM)
Ammonium (t~mol/gfwt)
Glutamine (~mol/gfwt)
a Glutamine synthetase (synthetase) (nmol/m/mg protein)
Ammonium chloride Ammonium q- Glutamate Ammonium q- Glutamine Potassium nitrate Nitrate q- Glutamate Nitrate -}- Glutamine
25.3 20.6 22.4 0.5 0.5 1.0
2.4 24.3 24.6 0.6 2.1 24.8
56 33 33 98 70 46
Synthetasc activity measured by the in vitro procedure. For other details see Table 1. adapted plants (40%), (Fig. 2). I n contrast to this differential response to glutamate, the addition of 5 mM glutamine is effective in reducing the glutamine synthetase level of both nitrate and ammonium adapted plants to similar extents (50% and 40% respectively), (Fig. 3). The effects of various concentrations of glutamate and glutamine are similar whether monitored b y the transferase or synthetase activity of the enzyme. I n plants grown on glutamine and nitrate there is a marked reduction in glutamine synthetase level in comparison to nitrate alone, and this is associated with a marked increase in the pool of glutamine, but no increase in the ammonium pool (Table 2). The addition of either glutamate or glutamine to ammonium adapted plants brings about a reduction in glutamine synthetase level, an increase in the glutamine pool and a reduction in the ammonium pool. The activity of plants grown on these combinations of nitrogen source is 25 % of the activity of y-aminobutyrate grown plants. A slight decrease in enzyme level occurs on the addition of glutamate to nitrate grown plants, and this is associated with a small increase in the pool of glutamine. These results are consistent with the idea t h a t the glutamine synthetase level is reduced in response to an increase in the glutamine pool. The changes in the pools of ammonium and glutamine with these combinations of nitrogen source support the concept of glutamine accumulation being restricted b y the availability of both glutamate and ammonium and these results suggest t h a t the effects of ammonium ions on the glutamine synthetase level are indirect, resulting from a higher pool of glutamine. Further evidence to support the idea t h a t glutamine synthetase is regulated b y the glutamine pool comes from the relationship between the apparent steadystate rate of glutamine synthetase synthesis, and the intracellular concentrations of ammonium ions and glutamine in plants grown on different nitrogen sources (Figs. 4 and 5). The apparent rate of enzyme synthesis has been calculated from its specific activity and growth rate. Such a means of describing the steady-state enzyme level is desirable since extractable protein, as a growth parameter, avoids complications arising from the gross morphological changes which occur on certain nitrogen sources in association with reduced growth rates.
Control of Glutamine Synthetase Level
207
3~ 2.5 /"2
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n .,+ [m.] Fig. 4. Relationship between the intracellular concentration of ammonium (~mol/ml of cell water), and the apparent steady-state rate of synthesis of glutamine synthetase [synthetase activity (nmol/m/mg protein)/Doubling time (h)], of plants adapted to different nitrogen sources supplied at 5 raM. Alanine (1), y-Aminobutyrate (2), Amino acid mixture 1 (3), Ammonium chloride (4), Ammonium nitrate (5), Ammonium~Glutamate (6), Ammonium~ Glutamine (7), Asparagine (8), Aspartate (9), Glutamate (10), Glutamine (11), Glycine (12), Potassium nitrate (13), Nitrate+Glutamate (14), Nitrate-[-Glutamine (15) 1 See Table 1 for final concentration of Amino acid mixture. For other details see Table 1.
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110 115 2=0 Glutamine [mm]
215
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Fig. 5. Relationship between the intracetlular concentration of glutamine, and the apparent steady-state rate of synthesis of glutamine synthetase of plants adapted to different nitrogen sources supplied at 5 raM. Units and symbols as for Fig. 4
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Activity
Fig. 6. Relationship between transferase and synthetase activities of glutamine synthetase of plants adapted to various nitrogen sources. The points shown are taken from Table 1 and Figs. 2 and 3. The line shown is the line of best fit, as determined by least square regression analysis of y on x: y = --0.1886+0.02689 x --0.00007x 2. r = 0.9831. S.E. = 0.0713
I n making the calculation of the apparent rate of enzyme synthesis it has been assumed that the specific activity of the enzyme remains constant during steady-state growth, and that the turnover of the enzyme is negligible. The intracellular metabolite concentrations have been determined from analyses of the soluble nitrogen pools and have been calculated on the basis of tissue water content rather than fresh weight. I n making this calculation it has been assumed that these metabolites are uniformly distributed within the cell. I t is evident from the results of Fig. 4 that there is no obvious relationship between the apparent rate of enzyme synthesis and the concentration of ammonium ions. Although in general the rate of synthesis falls with respect to ammonium concentration, it is clear that on some media, where the pool of ammonia is low, there is a low apparent rate of enzyme synthesis. There does however appear to be a much more clear cut relationship between the rate of enzyme synthesis and the concentration of the glutamine pool (Fig. 5). As the concentration of glutamine increases from 0.5 to 5 mM there is a marked decrease in the apparent rate of enzyme synthesis. At concentrations above 5 mM glutamine, the decline in this rate is much less pronounced. These results then suggest that the level of glutamine synthetase in the higher plant L e m n a m i n o r is subject to negative control by the endogenous pool of glutamine. I t has been suggested that this control is operational on both the transferase and synthetase activities to similar extents. A more critical appraisal of the relationship between the two activities is shown in Fig. 6 where the transferase
Control of Glutamine Synthetase Level
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and synthetase activities of Table 1 and Figs. 2 and 3 are correlated. It is clear from these results that the transferase: synthetase ratio is not a fixed parameter, the best fitting relationship is quadratic rather than linear. These results suggest that the control of glutamine synthetase by glutamine may involve slight changes in the transferase :synthetase ratio. The nature of these changes are, at present, not fully understood. Discussion
Stebbing (1974) has reviewed the evidence for the existence and properties of 'regulatory' control mechanisms (mechanisms that act so as to regulate the synthesis of macromolecular precursors when the regulatory metabolites are absent from the growth medium), and their relationship to ' adaptive' mechanisms (mechanisms that have been demonstrated only by the addition of the regulatory metabolites to the medium). Stebbing concludes that adaptive and regulatory controls may represent functionally different systems of control. It is clear therefore that adaptive responses in enzyme level to exogenously supplied metabolites can only be interpreted as having a true regulatory function if an appropriate correlation between enzyme level and the endogenous metabolite pool under consideration can be demonstrated. Moreover, this correlation should hold in a range of adaptive situations in the absence of the presumed regulatory metabolite from the growth medium. The results described in this paper suggest that the change in glutamine synthetase level in response to either exogenously supplied glutamine or variations in the endogenous pool of glutamine has such a regulatory function in Lemna. This is similar to the findings of Ferguson and Sims (1974a), where there is a negative correlation between the rate of glutamine synthetase synthesis and the pool of glutamine in the yeast Candida utilis. This relationship in Lemna is most evident over the range 0.5 to 5 mM. The failure of higher in vivo glutamine concentrations to further reduce the apparent rate of glutamine synthetase synthesis could be explained in two ways. Firstly it could be that there is compartmentalisation of the glutamine into seperate pools, only one of which is effective in modulating enzyme level. Alternatively, it might be that there is compartmentalisation of the enzyme or that there are functionally distinct isoenzymes. The possibility of enzyme compartmentalisation is supported by studies of its intracellular distribution, since at least a proportion of the enzyme appears to be located in the chloroplasts (Haystead, 1973; Miflin, 1974). At present we have no information of the possible existence of isoenzymes but in view of the different functions of glutamine it is possible that there might be two forms of the enzyme, one associated with the synthesis of glutamine for metabolic functions, the other providing glutamine for storage and transport of nitrogen, with only the former isoenzyme being regulated. The physiological significance of this control over glutamine synthetase level may lie in the relationship between glutamine and glutamate synthase. In the scheme proposed by Lea and Miflin (1974) it is suggested that it is through the combined operation of glutamine synthetase and glutamate synthase that ammonia is assimilated in higher plants. In this scheme the first enzyme of ammonia
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assimilation is glutamine synthetase, thus modulation of glutamine synthesis via the regulation of glutamine synthetase level could function to control the rate of glutamate synthesis as well as t h a t of glutamine. The simplest mechanism which could account for the reduction in the level of glutamine synthetase in response to increases in the glutamine pool is t h a t glutamine represses the synthesis of the enzyme. A possible alternative is t h a t glutamine mediates some form of inactivation of the enzyme. The rapid inactivation of glutamine synthetase in E. coli (5~ecke and Holzer, 1966) involves marked changes in the transferase:synthetase ratio and it is possible t h a t the slight changes in this ratio in L e m n a are a reflection of a similar form of inactivation. I n Candida utilis subtle changes in the ratio occur during the glutamine mediated inactivation of glutamine synthetase (Sims et al., 1974), but these changes are not evident in the long term adaptive response after the initial inactivation events. The possibility of inactivation of glutamine synthetase in L e m n a might therefore be better investigated in situations other than the longterm adaptive response described here. However, whatever the exact mechanism involved in the regulation of the glutamine synthetase level in L e m n a minor, it is clear t h a t in this organism there is control operating over glutamine synthesis by a mechanism other than feedback inhibition. This has considerable significance in view of severM reports t h a t feedback inhibition rather t h a n end product repression is the mechanism for the control of amino acid biosynthesis iil higher plants (see Miflin, 1973 for a discussion of this). Previous studies of nitrate assimilation in L e m n a minor (Stewart, 1972b; Orebamjo and Stewart, 1975) have established t h a t nitrate reductase is subject to end product repression. This, together with the control of glutamine synthetase level b y glutamine reported here, suggests the possibility t h a t in this organism end product repression m a y be an important form of control of both the assimilation and intermediary metabolism of nitrogen. D.R. was supported by an S.R.C. Studentship and G.A.R. by a British Council Technical Training Award.
References Brown, C. M., Mac-Donald, D. S., Meers, J. L. : Physiological aspects of microbial inorganic nitrogen metabolism. Advanc. Microbiol. Physiol. 11, 1-45 (1974) Dharmawardene, M. W. N., ttaystead, A., Stewart, W. D. P.: Glutamine synthetase of the nitrogen fixing alga Anaebaena cylindrica. Arch. Mikrobiol. 90, 281-296 (1973) Dougall, D. K. : Evidence for the presence of glutamate synthase in carrot cell cultures. Biochem. biophys. Res. Commun. 58, 639-646 (1974) Ferguson, A. R., Sims, A. P. : Inactivation in vivo of glutamine synthetase and NAD-specific glutamate dehydrogenase; its role in the regulation of glutamine synthesis in yeasts. J. gen. Microbiol. 69, 423-427 (1971) Ferguson, A.R., Sims, A.P.: The regulation of glutamine metabolism in Candida utilis: the role of glutamine in the control of glutamine synthetasc. J. gen. Microbiol. 80, 159-171 (1974a) Ferguson, A.R., Sims, A.P.: The regulation of glutamine metabolism in Candida utilis: the inactivation of glutamine synthetase. J. gen. Microbiol. 80, 173-185 (1974b) Gancedo, C., ttolzer, H. : Enzymatic inactivation of glutamine synthetase in Enterobacteriaceae. Europ. J. Biochem. 4, 190-192 (1968)
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Haystead, A.: Glutamine synthetase in the chloroplasts of Vicia [aba. Planta (Berl.) 111, 271-274 (1973) Kingdon, H. S. : Feedback inhibition of glutamine synthetase from green pea seeds. Arch. Biochem. 163, 429-431 (1974) ](ohlaw, G., Dragert, W., Holzer, H.: Parallel-Repression der Synthese von GlutaminSynthetase und DPN-abhgngiger Glutamatdehydrogenase in Hefe. Biochem. Z. 341, 224-238 (1965) Lea, P. J., 2VIiflin, B. J. : An alternative route for nitrogen assimilation in higher plants. Nature (Lond.) 251, 614-616 (1974) ~ecke, D., Holzer, H. : Repression und Inaktivicrung yon Glutamin-Synthetase in Escherichia coli durch NH +. Biochim. biophys. Acta (Amst.) 122, 341-351 (1966) Meers, J. L., Tempest., D. W. : Regulation of glutamine synthetase synthesis in some gramnegative bacteria. Biochem. J. 119, 603-605 (1970) Miflin, B. J. : Amino acid biosynthesis and its control in plants. In: Biosynthesis and its control in plants, ed. Milborrow, B. V., p. 49-68. London and New York: Acedemic Press 1973 Miflin, B. J.: The location of nitrate reductase and other enzymes related to amino acid biosynthesis in the plastids of roots and leaves. Plant Physiol. 54, 550-552 (1974) Orebamjo, T. 0., Stewart, G.R.: Some characteristics of nitrate reductase induction in Lemna minor L. Planta (Berl.) 117, 1-10 (1974) Orebamjo, T.O., Stewart, G. 1~.: Ammonium repression of nitrate reductase in Lemna minor L. Planta (Berl.) 122, 27-36 (1975) Pate, J. S. : Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem. 5, 109-119 (1973) Pateman, J. A. : Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms. Biochem. J. 115, 769-775 (1970) Ravel, J.M., Humphreys, J. S., Shire, W. : Control of glutamine synthesis in Lactobacillus arabinosus. Arch. Biochem. 111, 720-726 (1965) t~ebello, J. L. Strauss, N. : Regulation of glutamine synthetase in Bacillus subtilis. J. Bact. 98, 683-688 (1969) Rhodes, D., Stewart, G. R. : A procedure for the in vivo determination of enzyme activity in higher plant tissue. Planta (Berl.) 118, 133-144 (1974) Shapiro, B. M., Stadtman, E. R. : The regulation of glutamine synthesis in micro-organisms. Ann. Rev. Microbiol. 24, 501-524 (1970) Sims, A. P., Toone, J., Box, V.: The regulation of glutamine metabolism in Candida utilis: Mechanisms of control of glutamine synthetase. J. gen. Microbiol. 84, 149-162 (1974) Stebbing, N. : Precursor pools and endogenous control of enzyme synthesis and activity in biosynthetic pathways. Bact. Rev. 88, 1-28 (1974) Stewart, G. R. : The regulation of nitrite reductase level in Lemna minor L. J. exp. Bot. 23, 171-183 (1972a) Stewart, G. R. : End product repression of nitrate reductase in Lemna minor L. Symp. Biol. Hung. 13, 127-135 (1972b) Stewart, G. 1%., Lee, J. A., Orebamjo, T. O. : Nitrogen metabolism of halophytcs. II. Nitrate availability and utilisation. New Phytologist 72, 539-546 (1973) Streeter, J. G. : I n vivo and in vit~v studies on asparagine biosynthesis in soybean seedlings. Arch. Biochem. lgT, 613-624 (1973) Widholm, J.M.: Evidence for compartmentation of tryptophan in cultured plant tissues: Free tryptophan levels and inhibition of anthranilate synthetase. Physiol. Plant. 30, 323 326 (1974)
The Control of Glutamine Synthetase Level in Lemna minor L. David Rhodes, G. A. Rendon, and G. R. Stewart Department of Botany, The University, Manchester. M13 9PL, U.K. Received 10 may; accepted 21 May 1975 Summary. The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mlV[ ammonia and 5 mM glutamine as nitrogen source, lead to a 4-5 fold reduction of the maximum activity measurable on 5 mM y-aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutaminc. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.
Introduction The conversion of glutamic acid to glutamine is generally regarded as a key step in the intermediary nitrogen metabolism of micro-organisms (see e.g. Brown et al., 1974). The reason for this lies in the fact t h a t glutamine can serve as a nitrogen donor in the biosynthesis of several nitrogenous metabolites which are precursors of proteins and nucleic acids. I n view of this key role of glutamine it is not surprising t h a t the enzyme catalysing its synthesis is controlled b y a variety of complex regulatory mechanisms. The glutamine synthetase of Escherichia coli is in fact regulated b y several mechanisms including repression and derepression of its synthesis, cumulative feedback inhibition and rapid reversible inactivation (Shapiro and Stadtman, 1970). Repression of glutamine synthetase has been reported in other microorganisms, including Anabaena cylindrica (Dharmawardene et al., 1973). Bacillus subtilis (Rebello and Strauss, 1969), Candida utilis (Ferguson and Sims, 1974a), KlcbsieUa aerogenes (Meers and Tempest, 1970), Lactobacillus arabinosus (Ravel et al., 1965), Neurospora crassa and Aspergillus nidulans (Pateman, 1970) and in Saccharomyces cerevisiae (Kohlaw et al., 1965). I t is often considered t h a t ammonium is the co-repressor of enzyme synthesis (see Shapiro and Stadtman, 1970), although glutamine appears to have this role in Lactobacillus and Candida. I n contrast to repression control, the rapid inactivation by adenylation of the enzyme, first described in E. coli, appears to be restricted to certain members of the Gram-negative bacteria (Gancedo and ttolzer, 1968). However an alternative mechanism of rapid inactivation has been reported in several species of yeast (Ferguson and Sims, 1971). I n higher plants less is known of the metabolic function of glutamine although it acts as a nitrogen donor in the biosynthesis of asparagine (Streeter, 1973) 1 Planta (Berl.)
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a n d t r y p t o p h a n e (Widholm, 1974). R e c e n t l y a more stategic role for g l u t a m i n e i n higher p l a n t s has emerged with t h e discovery of a g l u t a m i n e d e p e n d e n t g l u t a m a t e s y n t h a s e (Dougall, 1974; Lea a n d Miflin, 1974). I n a d d i t i o n to these metabolic functions, g l u t a m i n e is i m p o r t a n t i n the storage a n d t r a n s p o r t of n i t r o g e n i n higher p l a n t s (Pate, 1973). A l t h o u g h the g l u t a m i n e s y n t h e t a s e (E.C. 6.3.1.2) of higher p l a n t s appears to be sensitive to a form of feedback i n h i b i t i o n similar to t h a t i n bacteria (ttaystead, 1973; K i n g d o n , 1974; Rhodes a n d Stewart, 1974), there is a lack of i n f o r m a t i o n available on the possible r e g u l a t i o n of its synthesis. W i t h these considerations i n m i n d , the regulation of g l u t a m i n e s y n t h e t a s e level i n the higher p l a n t L e m n a m i n o r has been studied.
Materials and Methods Organism and Growth Conditions. The strain of Lemna minor L. and the basal growth medium employed in the present study were those described previously (Stewart, 1972a). Plants were grown at 26~177176 under conditions of continuous illumination (30001ux). With the exception of glutamine, which was sterilised by millipore filtration, all other nitrogen sources were added to the medium immediately prior to autoclaving. The medium referred to as amino acid mixture was based on the composition of the pool of free amino acids in Lemna and contained the following concentrations of amino acids: alanine 0.37 raM, arginine 0.05 mM, aspartie 0.48 raM, cystine 0.005 raM, glutamic 0.86 raM, glycine 0.33 raM, histidene 0.025mM, isoleueine 0.13raM, leueine 0.14mM, Lysine 0.05raM, methionine 0.01 mi~, phenylalanine 0.045 raM, proline 0.03 mM, serine 0.58 raM, threonine 0.14 mM, tryptophane 0.01 raM, tyrosine 0.04 mM, and valine 0.06 mlVI. Growth Studies. Growth rates were determined using 100 ml of the appropriate medium with an initial innoculum of 10-15 fronds. Frond number was counted daily and the time for frond number to double was determined by regression analysis of log frond number versus time. Ammonia and Amino Acid Pool Analyses. These were extracted and determined as described previously (Orebamjo and Stewart, 1974). Enzyme Extraction and Assay. The enzyme extraction procedure was that described previously by Stewart (1972a). The optimum extraction buffer for the determination of glutamine synthetase activity was 0.05 M Imidazole-HC1, pI-I 7.2, containing 0.5 mM EDTA and 1.0 mM dithiothreitol. The reaction mixture for the determination of synthetase activity contained: 36 fzmol ATP, 90 tzmol MgS04, 12 Izmol hydroxylamine, 184 tzmol L-glutamate, and 100[zmol Imidazole-HC1. The final volume of the reaction mixture was 2 ml and had a pI-I of 7.2. The reaction was initiated by the addition of enzyme extract and was incubated at 30~ for 30 rain. The reaction mixture for the determination of the transferase activity contained: 0.34 Izmol ADP, 5 ~mol MnCI2, 34 Izmol hydroxylamine, 130 tzmol L-glutamine, 66 ~mol sodium arsenate and 200 tzmol Tris-acetate, pI:i 6.4 (final volume 2 ml). The reaction was initiated by the addition of enzyme extract and was incubated at 30~C for 10 rain. tIydroxymate was determined by the addition of ferric chloride reagent (Ferguson and Sims, 1971). Glutamyl Hydroxymate was Used as the Standard. Glutamine synthetase activity was determined in vivo as described by I~hodes and Stewart (1974). Plants were permeabilised by six cycles of freeze-thaw treatment followed by vacuum infiltration with the reaction mixture. The substrate concentrations were those used in the in vitro assays. Protein precipitated from the enzyme extracts with 10% triehloroacetic acid was determined as before (Stewart, 1972a). Total protein was determined by incubating plants in 60% ethanol overnight at room temperature to remove soluble Folin positive components. After a further 48 hrs. incubation in 0.72 M 1NaOH, the total protein content of the filtrate was determined as above. I n vitro enzyme activity is expressed as nmol or fzmolhydroxymate/m/mg extractable protein. I n vivo activity is expressed as tzmol/m/mg total protein. Total Nitrogen. The total nitrogen content of the plants was determined as described by Stewart et al. (1973).
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Table 1. Effect of nitrogen source on glutamine synthetase level, growth rate and total nitrogen content Glutamine synthetase (in vitro activity). Nitrogen source (5 re_M)
Synthetase (nmol/m/ mg protein)
Transferase (~zmol/m/ mg protein)
Doubling time (h)
Total nitrogen (% of dry wt)
Alanine y-Aminobutyrate Amino acid mixture a Ammonium chloride Ammonium nitrate Aspartate Asparagine Glutamate Glutamine Glycine Potassium nitrate
57 133 89 56 46 73 61 70 42 58 98
1.2 2.2 1.6 1.1 1.0 1.5 1.4 1.4 0.8 1.2 1.7
56.0 59.2 43.0 33.0 36.6 42.5 34.0 45.4 46.0 69.0 36.0
4.6 3.8 3.2 8.1 7.7 3.1 10.2 3.4 7.6 4.7 6.1
Results are the average of at least three separate determinations. Plants were adapted to different nitrogen sources through at least two sub-cultures before analysis. a Amino acid mixture total concentration 3.4 raM.
Results The specific activity of glutamine synthetase varies considerably depending on the source of nitrogen that L e m n a is grown on (Table l). Maximum activity of the enzyme was observed in plants grown on y-aminobutyrate and nitrate. The activity in plants grown on glutamine is some 3-4 fold lower than that in y-aminobutyric acid grown plants. Ammonium grown plants have a low specific activity of the enzyme compared with nitrate grown plants, but their activity is higher than that of glutamine grown plants. This pattern of variation in glutamine synthetase level is similar to that reported in many micro-organisms. The variation in specific activity with respect to nitrogen source is similar whether measured by the synthetase or transferase assay. There is no evidence for any marked change in the ratio of transferase to synthetasc activity. A comparison of enzyme level with either growth rate or nitrogen content indicates that there is no simple relationship between growth (or nitrogen status) and enzyme level. I n the observations described above, all nitrogen sources were present at a concentration of 5 raM. The relationship between enzyme level and the concentration of various nitrogen sources is shown in Fig. 1. The response to increasing concentrations of ammonium ions is striking, with there being a 50% reduction in specific activity as the concentration is increased from 0.1 to 5 raM. I n contrast, increasing the nitrate concentration has little effect on the glutamine synthetase level. Increasing the concentration of glutamate in the medium results in a small reduction in enzyme level. The pattern of response to increasing concentrations of the nitrogen source is similar whether measured by the in vitro or in vivo assay procedures.
204
D. Rhodes et al. A
B 1.0
0,7~
"~.
~>. I.0 u,i
0.25
i
i
r
x
1
2
3
4
J
Nitrogen Sourc9 rm M~
Fig. 1. Effect of nitrogen source concentration on the specific activity of glutamine synthetase (transferase) in vitro (A) and in vivo (B). Activity is expressed as ~mol/m/mg protein. Potassium nitrate (I), Glutamate (A), Ammonium chloride (e)
The results in Table 1 and Fig. 1 suggest that the glutamine synthetase level could be regulated by the endogenous pool of either glutamine or ammonium ions. In order to discriminate between these two possible effector molecules, plants have have been grown under conditions where the pools of ammonium and glutamine can be independently altered. Nitrate adapted plants are characterised by a low pool of both glutamine and ammonium (Orebamjo and Stewart, 1974). Under such conditions one can predict that the accumulation of glutamine is likely to be limited by the availability of ammonium ions. Ammonium adapted plants have a high pool of glutamine and ammonium (Orebamjo and Stewart, 1974) and under these conditions glutamate availability is more likely to limit the further accumulation of glutamine. I t is predictable then that the addition of glutamate to ammonium adapted plants should bring about an increase in the glutamine pool and a decrease in the pool of ammonium. The addition of glutamate to nitrate grown plants should not however result in any marked increase in the glutamine pool, due to the low availability of ammonium. The addition of glutamine to either ammonium or nitrate grown plants should bring about an increase in the pool of glutamine independent of the availability of ammonium ions. Accordingly, the response of nitrate and ammonium adapted plants to the addition of glutamate and glutamine have been determined. The effect of various concentrations of these nitrogen sources on the glutamine synthetase level of 5 mM nitrate and 5 mM ammonium grown plants is shown in Figs. 2 and 3. 5 mM glutamate brings about a 20% reduction in the enzyme level of nitrate adapted plants and a much more pronounced lowering of activity of ammonium
Control of Glutamine Synthetase Level
205
lOOE 1.5 ._>
9
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$ 0.5
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I
i
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Glutamate
Fig. 2. Effect of various concentrations of Glutamate on the specific activity of glutamine synthetase of 5 mM Nitrate grown plants (i), and 5 mM Ammonium grown plants (o). A: Synthetase activity (nmol/m/mg protein). B: Transferase activity (~mol/m/mg protein). Activities were determined by the in vitro procedure
10C
A
2.~! 1.5
5O
~ 1.o-
Glutarnine
[mM]
Fig. 3. Effect of various concentrations of Glutamine on the specific activity of glutamine synthetase of 5 mM Nitrate grown plants (i), and 5 mM Ammonium grown plants(o). A: Synthetase activity (nmol/m/mg protein). B: Transferase activity (~mol/m/mg protein). Activities were determined by the in vitro procedure
206
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Table 2. Effect of nitrogen source on soluble nitrogen pool and glutamine synthetase level Nitrogen source (5 mM)
Ammonium (t~mol/gfwt)
Glutamine (~mol/gfwt)
a Glutamine synthetase (synthetase) (nmol/m/mg protein)
Ammonium chloride Ammonium q- Glutamate Ammonium q- Glutamine Potassium nitrate Nitrate q- Glutamate Nitrate -}- Glutamine
25.3 20.6 22.4 0.5 0.5 1.0
2.4 24.3 24.6 0.6 2.1 24.8
56 33 33 98 70 46
Synthetasc activity measured by the in vitro procedure. For other details see Table 1. adapted plants (40%), (Fig. 2). I n contrast to this differential response to glutamate, the addition of 5 mM glutamine is effective in reducing the glutamine synthetase level of both nitrate and ammonium adapted plants to similar extents (50% and 40% respectively), (Fig. 3). The effects of various concentrations of glutamate and glutamine are similar whether monitored b y the transferase or synthetase activity of the enzyme. I n plants grown on glutamine and nitrate there is a marked reduction in glutamine synthetase level in comparison to nitrate alone, and this is associated with a marked increase in the pool of glutamine, but no increase in the ammonium pool (Table 2). The addition of either glutamate or glutamine to ammonium adapted plants brings about a reduction in glutamine synthetase level, an increase in the glutamine pool and a reduction in the ammonium pool. The activity of plants grown on these combinations of nitrogen source is 25 % of the activity of y-aminobutyrate grown plants. A slight decrease in enzyme level occurs on the addition of glutamate to nitrate grown plants, and this is associated with a small increase in the pool of glutamine. These results are consistent with the idea t h a t the glutamine synthetase level is reduced in response to an increase in the glutamine pool. The changes in the pools of ammonium and glutamine with these combinations of nitrogen source support the concept of glutamine accumulation being restricted b y the availability of both glutamate and ammonium and these results suggest t h a t the effects of ammonium ions on the glutamine synthetase level are indirect, resulting from a higher pool of glutamine. Further evidence to support the idea t h a t glutamine synthetase is regulated b y the glutamine pool comes from the relationship between the apparent steadystate rate of glutamine synthetase synthesis, and the intracellular concentrations of ammonium ions and glutamine in plants grown on different nitrogen sources (Figs. 4 and 5). The apparent rate of enzyme synthesis has been calculated from its specific activity and growth rate. Such a means of describing the steady-state enzyme level is desirable since extractable protein, as a growth parameter, avoids complications arising from the gross morphological changes which occur on certain nitrogen sources in association with reduced growth rates.
Control of Glutamine Synthetase Level
207
3~ 2.5 /"2
2,0t e3 l/ .8 -~----, o9 :=
E
9
/ js
,F,
W' 32
.~
96 02
n .,+ [m.] Fig. 4. Relationship between the intracellular concentration of ammonium (~mol/ml of cell water), and the apparent steady-state rate of synthesis of glutamine synthetase [synthetase activity (nmol/m/mg protein)/Doubling time (h)], of plants adapted to different nitrogen sources supplied at 5 raM. Alanine (1), y-Aminobutyrate (2), Amino acid mixture 1 (3), Ammonium chloride (4), Ammonium nitrate (5), Ammonium~Glutamate (6), Ammonium~ Glutamine (7), Asparagine (8), Aspartate (9), Glutamate (10), Glutamine (11), Glycine (12), Potassium nitrate (13), Nitrate+Glutamate (14), Nitrate-[-Glutamine (15) 1 See Table 1 for final concentration of Amino acid mixture. For other details see Table 1.
3"0I~13 2"5f! 2.0 3 1.5 1 " 1.0
"12
u~
e15
el
- -
eT-96
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o
;
110 115 2=0 Glutamine [mm]
215
3=0
Fig. 5. Relationship between the intracetlular concentration of glutamine, and the apparent steady-state rate of synthesis of glutamine synthetase of plants adapted to different nitrogen sources supplied at 5 raM. Units and symbols as for Fig. 4
208
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1.6 >~ 1~ v < ff
1.(
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9
9 o~
.! !" ~176
0.2
i
Synthetase
I X 150
Activity
Fig. 6. Relationship between transferase and synthetase activities of glutamine synthetase of plants adapted to various nitrogen sources. The points shown are taken from Table 1 and Figs. 2 and 3. The line shown is the line of best fit, as determined by least square regression analysis of y on x: y = --0.1886+0.02689 x --0.00007x 2. r = 0.9831. S.E. = 0.0713
I n making the calculation of the apparent rate of enzyme synthesis it has been assumed that the specific activity of the enzyme remains constant during steady-state growth, and that the turnover of the enzyme is negligible. The intracellular metabolite concentrations have been determined from analyses of the soluble nitrogen pools and have been calculated on the basis of tissue water content rather than fresh weight. I n making this calculation it has been assumed that these metabolites are uniformly distributed within the cell. I t is evident from the results of Fig. 4 that there is no obvious relationship between the apparent rate of enzyme synthesis and the concentration of ammonium ions. Although in general the rate of synthesis falls with respect to ammonium concentration, it is clear that on some media, where the pool of ammonia is low, there is a low apparent rate of enzyme synthesis. There does however appear to be a much more clear cut relationship between the rate of enzyme synthesis and the concentration of the glutamine pool (Fig. 5). As the concentration of glutamine increases from 0.5 to 5 mM there is a marked decrease in the apparent rate of enzyme synthesis. At concentrations above 5 mM glutamine, the decline in this rate is much less pronounced. These results then suggest that the level of glutamine synthetase in the higher plant L e m n a m i n o r is subject to negative control by the endogenous pool of glutamine. I t has been suggested that this control is operational on both the transferase and synthetase activities to similar extents. A more critical appraisal of the relationship between the two activities is shown in Fig. 6 where the transferase
Control of Glutamine Synthetase Level
209
and synthetase activities of Table 1 and Figs. 2 and 3 are correlated. It is clear from these results that the transferase: synthetase ratio is not a fixed parameter, the best fitting relationship is quadratic rather than linear. These results suggest that the control of glutamine synthetase by glutamine may involve slight changes in the transferase :synthetase ratio. The nature of these changes are, at present, not fully understood. Discussion
Stebbing (1974) has reviewed the evidence for the existence and properties of 'regulatory' control mechanisms (mechanisms that act so as to regulate the synthesis of macromolecular precursors when the regulatory metabolites are absent from the growth medium), and their relationship to ' adaptive' mechanisms (mechanisms that have been demonstrated only by the addition of the regulatory metabolites to the medium). Stebbing concludes that adaptive and regulatory controls may represent functionally different systems of control. It is clear therefore that adaptive responses in enzyme level to exogenously supplied metabolites can only be interpreted as having a true regulatory function if an appropriate correlation between enzyme level and the endogenous metabolite pool under consideration can be demonstrated. Moreover, this correlation should hold in a range of adaptive situations in the absence of the presumed regulatory metabolite from the growth medium. The results described in this paper suggest that the change in glutamine synthetase level in response to either exogenously supplied glutamine or variations in the endogenous pool of glutamine has such a regulatory function in Lemna. This is similar to the findings of Ferguson and Sims (1974a), where there is a negative correlation between the rate of glutamine synthetase synthesis and the pool of glutamine in the yeast Candida utilis. This relationship in Lemna is most evident over the range 0.5 to 5 mM. The failure of higher in vivo glutamine concentrations to further reduce the apparent rate of glutamine synthetase synthesis could be explained in two ways. Firstly it could be that there is compartmentalisation of the glutamine into seperate pools, only one of which is effective in modulating enzyme level. Alternatively, it might be that there is compartmentalisation of the enzyme or that there are functionally distinct isoenzymes. The possibility of enzyme compartmentalisation is supported by studies of its intracellular distribution, since at least a proportion of the enzyme appears to be located in the chloroplasts (Haystead, 1973; Miflin, 1974). At present we have no information of the possible existence of isoenzymes but in view of the different functions of glutamine it is possible that there might be two forms of the enzyme, one associated with the synthesis of glutamine for metabolic functions, the other providing glutamine for storage and transport of nitrogen, with only the former isoenzyme being regulated. The physiological significance of this control over glutamine synthetase level may lie in the relationship between glutamine and glutamate synthase. In the scheme proposed by Lea and Miflin (1974) it is suggested that it is through the combined operation of glutamine synthetase and glutamate synthase that ammonia is assimilated in higher plants. In this scheme the first enzyme of ammonia
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assimilation is glutamine synthetase, thus modulation of glutamine synthesis via the regulation of glutamine synthetase level could function to control the rate of glutamate synthesis as well as t h a t of glutamine. The simplest mechanism which could account for the reduction in the level of glutamine synthetase in response to increases in the glutamine pool is t h a t glutamine represses the synthesis of the enzyme. A possible alternative is t h a t glutamine mediates some form of inactivation of the enzyme. The rapid inactivation of glutamine synthetase in E. coli (5~ecke and Holzer, 1966) involves marked changes in the transferase:synthetase ratio and it is possible t h a t the slight changes in this ratio in L e m n a are a reflection of a similar form of inactivation. I n Candida utilis subtle changes in the ratio occur during the glutamine mediated inactivation of glutamine synthetase (Sims et al., 1974), but these changes are not evident in the long term adaptive response after the initial inactivation events. The possibility of inactivation of glutamine synthetase in L e m n a might therefore be better investigated in situations other than the longterm adaptive response described here. However, whatever the exact mechanism involved in the regulation of the glutamine synthetase level in L e m n a minor, it is clear t h a t in this organism there is control operating over glutamine synthesis by a mechanism other than feedback inhibition. This has considerable significance in view of severM reports t h a t feedback inhibition rather t h a n end product repression is the mechanism for the control of amino acid biosynthesis iil higher plants (see Miflin, 1973 for a discussion of this). Previous studies of nitrate assimilation in L e m n a minor (Stewart, 1972b; Orebamjo and Stewart, 1975) have established t h a t nitrate reductase is subject to end product repression. This, together with the control of glutamine synthetase level b y glutamine reported here, suggests the possibility t h a t in this organism end product repression m a y be an important form of control of both the assimilation and intermediary metabolism of nitrogen. D.R. was supported by an S.R.C. Studentship and G.A.R. by a British Council Technical Training Award.
References Brown, C. M., Mac-Donald, D. S., Meers, J. L. : Physiological aspects of microbial inorganic nitrogen metabolism. Advanc. Microbiol. Physiol. 11, 1-45 (1974) Dharmawardene, M. W. N., ttaystead, A., Stewart, W. D. P.: Glutamine synthetase of the nitrogen fixing alga Anaebaena cylindrica. Arch. Mikrobiol. 90, 281-296 (1973) Dougall, D. K. : Evidence for the presence of glutamate synthase in carrot cell cultures. Biochem. biophys. Res. Commun. 58, 639-646 (1974) Ferguson, A. R., Sims, A. P. : Inactivation in vivo of glutamine synthetase and NAD-specific glutamate dehydrogenase; its role in the regulation of glutamine synthesis in yeasts. J. gen. Microbiol. 69, 423-427 (1971) Ferguson, A.R., Sims, A.P.: The regulation of glutamine metabolism in Candida utilis: the role of glutamine in the control of glutamine synthetasc. J. gen. Microbiol. 80, 159-171 (1974a) Ferguson, A.R., Sims, A.P.: The regulation of glutamine metabolism in Candida utilis: the inactivation of glutamine synthetase. J. gen. Microbiol. 80, 173-185 (1974b) Gancedo, C., ttolzer, H. : Enzymatic inactivation of glutamine synthetase in Enterobacteriaceae. Europ. J. Biochem. 4, 190-192 (1968)
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Haystead, A.: Glutamine synthetase in the chloroplasts of Vicia [aba. Planta (Berl.) 111, 271-274 (1973) Kingdon, H. S. : Feedback inhibition of glutamine synthetase from green pea seeds. Arch. Biochem. 163, 429-431 (1974) ](ohlaw, G., Dragert, W., Holzer, H.: Parallel-Repression der Synthese von GlutaminSynthetase und DPN-abhgngiger Glutamatdehydrogenase in Hefe. Biochem. Z. 341, 224-238 (1965) Lea, P. J., 2VIiflin, B. J. : An alternative route for nitrogen assimilation in higher plants. Nature (Lond.) 251, 614-616 (1974) ~ecke, D., Holzer, H. : Repression und Inaktivicrung yon Glutamin-Synthetase in Escherichia coli durch NH +. Biochim. biophys. Acta (Amst.) 122, 341-351 (1966) Meers, J. L., Tempest., D. W. : Regulation of glutamine synthetase synthesis in some gramnegative bacteria. Biochem. J. 119, 603-605 (1970) Miflin, B. J. : Amino acid biosynthesis and its control in plants. In: Biosynthesis and its control in plants, ed. Milborrow, B. V., p. 49-68. London and New York: Acedemic Press 1973 Miflin, B. J.: The location of nitrate reductase and other enzymes related to amino acid biosynthesis in the plastids of roots and leaves. Plant Physiol. 54, 550-552 (1974) Orebamjo, T. 0., Stewart, G.R.: Some characteristics of nitrate reductase induction in Lemna minor L. Planta (Berl.) 117, 1-10 (1974) Orebamjo, T.O., Stewart, G. 1~.: Ammonium repression of nitrate reductase in Lemna minor L. Planta (Berl.) 122, 27-36 (1975) Pate, J. S. : Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem. 5, 109-119 (1973) Pateman, J. A. : Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms. Biochem. J. 115, 769-775 (1970) Ravel, J.M., Humphreys, J. S., Shire, W. : Control of glutamine synthesis in Lactobacillus arabinosus. Arch. Biochem. 111, 720-726 (1965) t~ebello, J. L. Strauss, N. : Regulation of glutamine synthetase in Bacillus subtilis. J. Bact. 98, 683-688 (1969) Rhodes, D., Stewart, G. R. : A procedure for the in vivo determination of enzyme activity in higher plant tissue. Planta (Berl.) 118, 133-144 (1974) Shapiro, B. M., Stadtman, E. R. : The regulation of glutamine synthesis in micro-organisms. Ann. Rev. Microbiol. 24, 501-524 (1970) Sims, A. P., Toone, J., Box, V.: The regulation of glutamine metabolism in Candida utilis: Mechanisms of control of glutamine synthetase. J. gen. Microbiol. 84, 149-162 (1974) Stebbing, N. : Precursor pools and endogenous control of enzyme synthesis and activity in biosynthetic pathways. Bact. Rev. 88, 1-28 (1974) Stewart, G. R. : The regulation of nitrite reductase level in Lemna minor L. J. exp. Bot. 23, 171-183 (1972a) Stewart, G. R. : End product repression of nitrate reductase in Lemna minor L. Symp. Biol. Hung. 13, 127-135 (1972b) Stewart, G. 1%., Lee, J. A., Orebamjo, T. O. : Nitrogen metabolism of halophytcs. II. Nitrate availability and utilisation. New Phytologist 72, 539-546 (1973) Streeter, J. G. : I n vivo and in vit~v studies on asparagine biosynthesis in soybean seedlings. Arch. Biochem. lgT, 613-624 (1973) Widholm, J.M.: Evidence for compartmentation of tryptophan in cultured plant tissues: Free tryptophan levels and inhibition of anthranilate synthetase. Physiol. Plant. 30, 323 326 (1974)
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